SF 2S3 



.A* 



rz 




BULLETIN OF THE 



No. 202 




Contribution from the Bureau of Animal Industry, A. D. Melvin, Chief. 
May 12, 1915. 

(PROFESSIONAL PAPER.) 

THE ALCOHOL TEST IN RELATION TO MILK. 

By S. Henry Ayers, Bacteriologist, and William T. Johnson, Jr., Scientific Assistant, 

Dairy Division. 

INTRODUCTION. 

The alcohol test as generally used consists in the mixing of equal 
volumes of alcohol and milk. Usually 2 cubic centimeters of 68 per 
cent alcohol are added to 2 cubic centimeters of milk and shaken 
gently in a test tube. The test is considered positive when a precipi- 
tate is formed, or in other terms, when a coagulum is produced. 
When a positive test is obtained with fresh milk from a single cow or 
small herd, it is generally believed that it indicates an abnormal milk, 
due to physiological or pathological conditions in the cow. A positive 
test with market milk is supposed to indicate that changes have been 
produced in the milk as a result of bacterial fermentations. 

According to Fleischmann (ll) 1 the first account of the alcohol 
test was published by Martinn in 1890 in the Deutsche (Berliner) 
Molkerei Zeitung. It is stated that Martinn used 68 per cent alcohol 
with equal parts of milk. Hoft (13) in 1898 used the alcohol test to 
give an idea of the acidity of milk. He found that the higher the acidity 
the greater the amount of coagulation by alcohol. In the same year 
Petri and Maaszen (24) made use of the alcohol test to determine the 
quality of pasteurized milk, and Weber (31) in 1900 studied the alcohol 
test in relation to the so-called sterilized milk. 

Since 1900 numerous investigators, mostly in Europe, have studied 
the alcohol test. Of those who have worked with this test Morres is 
probably its most ardent supporter. He strongly advocates the 
alcohol test in combination with the alizarin test, which he calls the 
alizarol test. This test will be described later. Morres and the 
other advocates of the alcohol test claim that it is of great value, 
since it affords a simple and quick means of determining the condition 
and keeping quality of milk. 

In this country the alcohol test is used by only one large company 
which manufactures milk powder. Any milk which shows a precipi- 

1 See list of citations to literature at end of bulletin. 
82832°— Bull. 202—15 1 



Monograph 



ograph 



rd 



2 BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 

tate when mixed with equal volumes of 75 per cent alcohol is rejected 
by this company. We are not aware that any practical use of the test 
is made by any one else in America. In Europe the alcohol test is 
more generally used, but we are unable to state to what extent the 
test is employed at present, although Farrington and Woll (9) say 
that in European creameries and city milk depots the alcohol test is 
often applied to every can of milk received; milk that is sufficiently 
sour to be noticed by the taste will coagulate when mixed with an 
equal volume of 70 per cent alcohol. 

The Berlin police regulation of 1902 (32) regarding the sale of milk 
and cream required that cow's milk coming from a distance must, 
at the time of delivery to the consumer, stand without coagulation 
the cooking or alcohol test (mixture of 70 per cent alcohol by volume 
with equal parts of milk). According to Devarda and Weich (6), 
only fresh milk, which shows no precipitate or only a very fine coagu- 
lation with the alcohol test, is accepted in the Vienna market. 

OBJECT OF THIS WORK. 

The principal object of this work was to determine the practical 

value of the alcohol test as a test for the quality of market milk. As 

incidental to our primary object, it was our purpose to determine 

some of the causes for the precipitation or coagulation of milk by 

alcohol. 

METHOD OF MAKING THE ALCOHOL TEST. 

In our work we have used the single alcohol test; that is to say, a 
mixture of equal volumes of alcohol and milk. A few investigators 
have used the double alcohol test, in which two parts by volume of 
alcohol are mixed with one part of milk. In general equal volumes 
of 68 per cent alcohol and milk are mixed for the test, but in our 
work 75 per cent, 68 per cent, and 44 per cent alcohol were used. 
Three tests were made on each sample of milk, 2 c. c. of alcohol 
being mixed with 2 c. c. of milk in a test tube. The milk was always 
at a temperature of from 15° to 20° C. After adding the milk to the 
alcohol the tube was shaken and examined for the appearance of a 
precipitate. The precipitate appears as flakes the size of which were 
recorded as follows: VS for very small, S for small, M for medium- 
sized, and L for large. 

The different percentages of alcohol were obtained by diluting a 
high grade of absolute alcohol with distilled water. Reiss (27) has 
shown that alcohol should always be tested for acid before using in 
the alcohol test, as acetic acid sometimes found in the alcohol may 
make the milk sufficiently acid to cause a coagulation with alcohol. 
The acidity was determined by titrating 10 c. c. of milk with N/10 
NaOH, and is expressed throughout this paper as per cent of normal acid. 

Any special methods employed in this work will be discussed when 
mentioned in the text. 

d; of j> 

MAY • IS 



THE ALCOHOL TEST IN EELATION TO MILK. 3 

THE ALCOHOL TEST IN RELATION TO FRESH MILK FROM A SINGLE COW 

OR HERD. 

While reviewing the literature on the alcohol test it became evi- 
dent that the value of the test must be considered from two stand- 
points: First, its relation to fresh milk from a single cow or small 
herd, and, second, its relation to mixed market milk. Although 
our work on this subject deals principally with the relation of the 
alcohol test to mixed market milk, we feel justified, after a careful 
survey of the literature, first in briefly discussing the test in its rela- 
tion to fresh -milk from a single cow or herd. 

In the consideration of fresh milk from a single, normal cow we 
must omit the changes in milk due to bacterial growth and the in- 
fluences of the changes on the alcohol test. The changes as a result 
of bacterial activities are of greater importance in the relation of the 
alcohol test to the mixed market milk and will be discussed later. 

It is evident from the results of other investigators and from our 
own tests on milk from a few cows that fresh, normal milk occasion- 
ally coagulates with 68 or 70 per cent alcohol when mixed in equal 
volumes. Henkel (12) found, after an examination of more than 
1,600 samples of milk from a single cow, that 6 showed a coagulation 
with 68 or 70 per cent alcohol. This is a very low percentage of 
positive results and he concluded that, generally speaking, the milk 
of a single animal does not coagulate with 68 or 70 per cent alcohol. 
After an extensive study of the alcohol test Auzinger (2) concluded 
that the alcohol coagulation of fresh single milk is not so rare as 
Henkel had observed. Auzinger (2) also found great fluctuations in 
the alcohol test (70 per cent) with milk from single cows. Occa- 
sionally milk from the same cow gave a positive test in the morning 
and not in the evening, or vice versa. The test might be positive 
one day and not the next, but might reappear on the third day. 
Sometimes he found that the first and last milk from a single cow 
showed fluctuations in the alcohol test. Auzinger also found that 
milk from single quarters may coagulate with alcohol independently 
of the other quarters, although these cases were rare. He concludes 
that the alcohol test in normal milk from a single cow is independent 
of the acidity and when the test is positive it is caused by a change 
in the milk salts, especially the calcium, in their relation to the milk 
proteids. His opinion as to the reason for the occasional coagula- 
tion of fresh, normal milk is strengthened by one of his experiments, 
in which calcium phosphate was fed to a cow. It was found that the 
milk from this cow coagulated with a smaller volume of alcohol or 
with a lower percentage of alcohol than did the normal milk. 

When fresh, normal milk from a single cow coagulates with 68 per 
cent alcohol it is evidently due to some slight change in the com- 
position of the milk. What the exact changes are it is impossible at 
present to state. 



4 BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 

When we speak of fresh, normal milk we mean fresh milk from a 
healthy cow in the middle of the period of lactation. Milk in the early 
period of lactation, that is, colostrum milk, or milk taken late in the 
lactation period — "old" milk, as it is sometimes called — usually 
coagulates with the alcohol test. Henkel (12), Metzger (17), and 
also Auzinger (2), found that the milk from a cow in the first of the 
lactation period, while apparently normal, may show a positive alcohol 
test at irregular intervals. Auzinger (2) believes that the high albu- 
men and globulin content of colostrum milk and the calcium salts are 
responsible for the positive alcohol reaction. 

EFFECT OF COLOSTRUM AND OF "OLD" MILK ON THE ALCOHOL TEST. 

In Table 1 are shown the results of the alcohol tests which we have 
made on colostrum milk from two cows. Three tests were made, 
using 75, 68, and 44 per cent alcohol. The results show clearly that 
colostrum milk gives a positive alcohol test and that the stronger the 
alcohol the longer the test will be positive. It will be noticed that the 
milk from cow 16 gave a positive test with 68 per cent alcohol for 24 
days, although the acidity was low after the fourth day. It is evi- 
dent from these results and from those obtained by other investi- 
gators that the coagulation of milk in the first of the lactation period 
by alcohol is largely independent of acidity. 

Table 1. — Alcohol tests with colostrum milk. 











Alcohol test. 




Cow 
No. 


Days after 
calving. 


Acidity. 




















75 per cent. 


68 per cent. 


44 per cent. 


4 


2 


2.61 


i+L 


+L 


+VS 




3 


2.45 


+L 


+L 


+VS 




4 


2.25 


+L 


+M 


— 




5 


1.87 


+L 


+s 


— 




6 


1.80 


+M 


— 


— 




8 


1.50 


+M 


— 


— 




9 


1.70 


+M 


— 


— 




10 


1.55 


— 


— 


— 




11 


1.52 


+s 


— 


— 




12 


1.50 


+M 


— 


— 




13 


1.31 


+s 


— 


— 




19 


1.35 




— 


— 




21 


2.10 


— 


— 


— 




22 


1.12 


_ 





— 


16 


1 


2.40 


+L 


+ L 


— 




2 


2.20 


+L 


+s 


— 




3 


2.70 


+M 


+M 


— 




4 


2.26 


+L 


+s 


— 




5 


1.60 


+ M 


+s 


— 




6 


1.84 


+ M 


+s 


— 




13 


1.36 


+L 


+M 


— 




15 


1.65 


+M 


+s 


— 




16 


1.53 


+M 


+M 


_ 




19 


1.57 


+M 


+s 


— 




22 


1.70 


+M 


+s 


— 




23 


1.60 


+ M 


+s 


— 




24 


1.50 


+ M 


+s 


— 




25 


1.45 


+M 


— 


— 



1 In this and succeeding tables the initial letters denoting the degree of the positive ( + ) tests signify: 
L, large flakes; M, medium flakes; S, small flakes; and VS, very small flakes. Minus sign (—) signifies 
negative test. 



THE ALCOHOL TEST IN RELATION TO MILK. 



In order to determine whether or not the alcohol test would be 
positive in a mixed colostrum and normal milk, one experiment was 
performed. Colostrum milk from two cows 24 hours after calving 
was mixed in various proportions with fresh, normal milk which gave 
a negative alcohol test. The results of this experiment, in Table 2, 
show that from 80 to 90 per cent of colostrum milk had to be mixed with 
normal milk in order to cause a positive test with 68 per cent alcohol. 
When 75 per cent alcohol was used the test was positive with as low 
as 25 per cent of colostrum milk from cow 5, but when colostrum 
milk from cow 16 was used, a mixture of 80 per cent was required to 
give a positive reaction with 75 per cent alcohol. It seems evident 
from these results that the mixing of colostrum and normal milk 
would not cause a positive alcohol test unless a very large percentage 
of the milk were colostrum milk. 

Table 2. — The alcohol test with a mixture of normal and colostrum milk. 



Colostrum 
milk from 
cow No. 


Percentage 

of 

normal 

milk. 


Percentage 

of 

colostrum 

milk. 


Alcohol test. 


75 per cent. 


68 per cent. 


44 per cent. 


5 
16 


10 
20 
25 

50 
75 
90 
10 
20 
25 


90 
80 
75 
50 
25 
10 
90 
80 
75 


>+L 
+M 

+M 

+s 

+ S 
+ M 

+s 


+ L 
+VS 

+ M 


- 



1 See footnote under Table 1. 

Having discussed the relation of the alcohol test to colostrum milk, 
let us consider its relation to milk drawn at the last of the lactation 
period, or what is known as "old" milk. Several investigators have 
shown that " old" milk gives a positive alcohol test. It is well known 
that milk changes in composition toward the end of the lactation 
period, and it is undoubtedly these changes which cause the coagu- 
lation with alcohol. While no definite changes have been attributed 
to the positive alcohol reaction, it is believed by some to be due to 
the high content of solids (not fat). Henkel (12), however, found 
that this could not explain in all cases the coagulation by alcohol. 
Auzinger (2) believes that on account of the variation of solids (not 
fat) the alcohol test has no significance in milk from "old" milk 
cows. 

SUMMARY OF CAUSES FOR POSITIVE TESTS IN MILK OF SINGLE COWS. 

It is apparent that fresh milk from a single cow may occasionally 
give a positive alcohol reaction with 68 or 70 per cent of alcohol. 
Colostrum milk gives a positive reaction, and the same is true usu- 



6 BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 

ally of "old" milk — that is, milk from a cow in the last of its lac- 
tation period. 

The causes for a positive alcohol test may be summarized by the 
opinion of Ernst (8) who states that a positive alcohol test of fresh 
milk from a single cow indicates a physiological or severe patho- 
logical condition of irritation of the milk glands. There is, how- 
ever, a difference in the opinions of various investigators as to the 
reaction of the alcohol test to pathological conditions of the udder. 
Ruhm (28) noticed the alcohol test in milk from cows with in- 
fected udders. In some cases he found the test was positive during 
the infection and frequently a positive test was observed for three 
or four weeks later when the milk had a normal appearance and 
taste. He points out that in udder infection the milk may vary in 
many ways, and in consequence the alcohol test varies. Auzinger 
found that there was no relation between streptococci in infected 
udders and the alcohol test and that a positive test is produced 
through chemical changes in the secretions. Rullmann and Tromms- 
dorff (29) also observed a positive alcohol reaction in milk from cows 
with infected udders, but according to these authors the alcohol test 
shows no definite relation to the leucocyte count. They point out 
that the variation in ash salts and high albumin content probably 
influences the alcohol test. Campbell (5) also believes that the alcohol 
test is of value in determining the diseased condition of the udder. 
Besides udder infection Auzinger (2) states that the general infections 
and infections of the vaginal canal may cause a positive alcohol test; 
also that milk from cows which have aborted may coagulate with 
alcohol. Metzger (17), however, after a study of the alcohol test 
with milk from sick cows concludes that the milk from them shows 
no relation between the acidity and alcohol test. According to this 
author fever had no influence on the acid and alcohol tests. There 
was no relation between tuberculosis of the animal and the alcohol 
test. When animals were very lean from disease the milk inclined 
toward coagulation with alcohol. Infectious inflammation of the 
vagina was without influence on the test. Infection of the uterus 
shows almost regularly with the alcohol test, but not without ex- 
ception. Metzger also found that there was no relation between the 
alcohol test and various forms of indigestion. He points out that 
the chief value of the test lies in its use for the freshness of milk. 

We have not had an opportunity to study the alcohol test in its 
relation to the milk from sick cows, but from a study of the literature 
on this subject we are inclined to believe that the alcohol test would 
be of but little value as a routine test of the milk from a single cow 
or from a small herd. If the alcohol test were used regularly to test 
fresh milk of single cows a positive reaction would indicate some 
change in the milk from normal. Subsequent examination of the 



THE ALCOHOL TEST IN" RELATION TO MILK. 7 

cow might reveal some pathological condition, or there might be 
some physiological reason for a slight variation in the composition 
of the milk. If the test were performed on the milk from a few cows 
a positive reaction might be caused, as Auzinger (3) believes, by the 
mixing of milk which is changed by physiological or pathological 
conditions with milk from normal cows. If there were a large per- 
centage of abnormal milk which gave an alcohol test with a coagula- 
tion with large flakes, the mixed milk might show a positive alcohol 
test in which the coagulation would be in the form of small flakes. 
When mixed milk from a large number of sources gives a positive 
alcohol test it must be interpreted in an entirely different manner, 
and this leads us to another phase of the subject. 

THE ALCOHOL TEST IN RELATION TO MARKET MILK. 

Since 1900 a considerable number of papers have appeared on the 
use of the alcohol test in its relation to market milk. According to 
Kirchner (15), Morres in 1905 showed that the alcohol test was of 
value for determining the keeping quality of milk and indicating its 
acidity. Reiss (26) in 1906 pointed out the practical value of the 
test, and Morres (18) again in 1909 showed the value of the alcohol 
test as a means of determining the keeping quality of milk. He added 
2 c.c. of milk to 2 c.c. of 68 per cent (by volume) alcohol, and states 
that if the milk coagulates with alcohol then decomposition has 
already started and the extent is shown by the size of the flakes. 
If the precipitate is in fine flakes then the acidity corresponds to 
4 degrees Soxhlet; however, the coagulation may not be due to an 
increase in acidity, but may be due to the action of rennet-forming 
bacteria. In later work Morres has combined the alcohol and 
alizarin tests. This will be discussed later. Morres considers that 
the coagulation of mixed market milk is due largely to the formation 
of acid or the action of rennet-forming bacteria or to a combination 
of both. Henkel (12) concludes from his work that the alcohol test 
does not afford a proper means for determining acidity, but that the 
value of the test lies in the fact that it gives a knowledge of the souring 
and other changes in the properties of milk or in variations from the 
normal properties which the acid test does not show. Other investi- 
gators believe that the alcohol test is of value only as a preliminary 
test. Fendler and Borkel (10) after a large number of tests to 
determine the relation of the acidity of milk to the alcohol test con- 
cluded that the double test with 70 per cent alcohol was not a proper 
criterion for the freshness of market milk, including infants' milk 
and superior grades of milk. They state that the double test using 
50 per cent alcohol is suitable as a preliminary test for food in- 
spectors, but the milk should be submitted to further tests. These 
authors also found that no consistent relation existed between the 



8 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



alcohol test and the acidity of milk. Rammstedt (25) also agrees 
with Fendler and Borkel, so far as he found, that no consistent 
relation existed between the alcohol test and the acidity of milk. 
He considers that the test gives preliminary knowledge of the hygienic 
quality of a milk. 

It is evident from the literature that in a mixed market milk the 
acidity plays a part in connection with the alcohol test, so that in 
considering the factors which influence the test we may first take up 
the question of acidity. 

THE INFLUENCE OF ACIDITY ON THE ALCOHOL TEST. 

In our first experiments the acidity of milk was raised by the 
addition of N/10 lactic acid. The results of two experiments recorded 
in Table 3 show that a very slight increase in the acidity of milk may 
cause a positive alcohol test with 75 per cent and 68 per cent alcohol, 
but a considerably higher acidity is required to cause a positive test 
with 44 per cent alcohol. 

These results show clearly that the alcohol test is sensitive to slight 
changes in acidity when these changes are produced by the addition 
of lactic acid. Since an increase in acidity will cause a positive 
alcohol test it is evident that the growth of acid-forming bacteria in 
milk will cause a positive test. 

Table 3. — Influence of acidity on the alcohol test. 



N/10 lactic 






Alcohol test. 




acid added 
to 50 c. c. 


Acidity. 














of milk. 




75 per cent. 


68 per cent. 


44 per cent. 


c. c. 



1.81 








0.5 


1.88 


— 


— 


— 


1.0 


1.94 


i + M 


+ M 


— 


3.0 


2.21 


+ L 


+L 


— 


3.5 


2.38 


+ L 


+ L 


— 


4.0 


2.47 


+ L 


+ L 


+M 





1.70 


— 


— 


— 


.5 


1.76 


+ M 


— 


— 


1.0 


1.84 


+ M 


+s 


— 


2.0 


2.00 


+ L 


+ L 


— 


3.0 


2.20 


+ L 


+ L 


— 


3.4 


2.25 


+ L 


+ L 


— 


3.5 


2.26 


+ L 


+.L 


+few VS 


4.0 


2.31 


+ L 


+ L 


+ few L 



i See footnote under Table 1. 

In order to determine the relation between the number of acid- 
forming bacteria, the acidity, and the alcohol test, two experiments 
were performed, using a pure culture of a lactic-acid-producing organ- 
ism. The culture was inoculated into sterile skim milk and incubated 
at 37° C. A bacterial count was made while the acidity and the 
alcohol test were determined at the same time. From the results 
shown in Table 4 it may be seen that in Experiment I the alcohol 
test was negative even after seven hours of incubation. At that time 



THE ALCOHOL TEST IN BELATION TO MILK. 9 

the acidity had increased from 1.98 to 2.14, and the bacteria from 
from 82,000 to 15,100,000 per cubic centimeter. It is interesting to 
note that an extensive multiplication of lactic-acid-forming bacteria 
may occur without causing a positive alcohol test. In the second 
experiment, also shown in Table 4, a heavier inoculation was used, and 
it will be seen that the milk at the beginning of the incubation period 
contained 480,000 bacteria per cubic centimeter. The 68 per cent 
alcohol test was not positive until the bacteria had increased to 
31,400,000 per cubic centimeter. 

These figures show that when a pure culture of lactic-acid-forming 
bacteria is grown in skim milk there must be a very great increase in 
order to produce acidity enough to cause a positive alcohol test. In 
these experiments there were no positive alcohol tests until the bac- 
teria had increased from less than 500,000 to over 16,000,000 per cubic 
centimeter. From these results it is apparent that the growth of 
acid-forming bacteria in milk may, through the formation of acid, 
cause a positive alcohol test. However, when there is sufficient acid 
produced to cause a coagulation with 68 per cent alcohol the number 
of acid-forming bacteria would be very high. 

Table 4. — Influence on tfie alcohol test of acid produced by the growth of a -pure culture of 

lactic-acid bacteria. 



Experi- 
ment No. 


Age of 
culture 
in hours. 


Bacteria 

per cubic 

centimeter. 


Acidity. 


Alcohol test. 


75 per cent. 


68 per cent. 


44 per cent. 


I 
II 



2 
4 
5 
6 
7 

2 
4 
5 
6 
7 


82,000 

113,000 

1,510,000 

4,300,000 

11,700,000 

15,100,000 

4S0,000 

1,060,000 

7,500,000 

16,100,000 

31,400,000 

46,000,000 


1.98 


+ L 
+ L 


+M 
+ L 


- 


2.06 
2.08 
2.09 
2.14 
1.94 


2.08 
2.08 
2.30 
2.47 



1 See footnote under Table 1. 



EFFECT OF PHOSPHATES. 



We have so far discussed in a general way the effect of increasing 
the acidity of milk by the addition of lactic acid and by the generation 
of the acid in milk. Since the acidity of milk when titrated with 
phenolphthalein is due partly to acid phosphates, it will be of interest 
to show the effect on the alcohol test of the increase in acidity by 
acid phosphates. In Table 5 are shown the results of a few tests, 
using sodium and potassium acid phosphate. Various amounts of 
a 5 per cent solution of these salts were added to 50 c. c. of milk. It 
will be seen from the table that when the acidity was increased by 
sodium acid phosphate from 2.15 to 3.33 the alcohol test with 75 



82832°— Bull. 202—15- 



10 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



per cent alcohol was positive. At an acidity of 4.27 the milk coagu- 
lated with 68 per cent alcohol but the flakes were very small. In 
order to cause a coagulation with 68 per cent alcohol with medium- 
sized flakes it was necessary to increase the acidity to 6.16. When 
potassium acid phosphate was used the results were about the same. 
These results show that it is possible by increasing the acidity of 
milk with acid phosphates to cause a coagulation with the alcohol 
test, but the acidity has to be increased to a high degree and there 
would never be enough acid phosphate in a mixed market milk for 
it to be entirely responsible for a positive alcohol test. 

Table 5. — Influence on the alcohol test of the addition of acid phosphates to milk. 



Sodium-acid phosphate. 


Potassium-acid phosphate. 


Amount 

of 5 
per cent 
solution 
of acid 
phos- 
phate 
added to 
50 c. c. of 
milk. 


Acid- 
ity. 


Alcohol test. 


Amount 

of 5 
per cent 
solution 
of acid 
phos- 
phate 
added to 
50 c. c. of 
milk. 


Acid- 
ity. 


Alcohol test. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


C.c. 

1 
2 
3 
5 
6 


2.15 
2.75 
3.33 
4.27 
5.50 
6.16 


i+M 

+ M 
+ M 
+ M 


+ VS 
+ VS 
+ M 


+VS 


C.c. 

1 
2 
3 
5 
6 


2.52 
3.13 
4.00 
5.20 
5.62 


I +M 
+ M 
+ M 
+ M 


+ VS 
+ VS 
+ M 


+ VS 



1 See footnote under Table 1. 

In some cases where we increased the acidity of milk by adding 
lactic acid it was noticed that a very slight increase in acidity caused 
a positive alcohol test. At other times the acidity had to be in- 
creased to a considerable extent before the milk coagulated with 
alcohol. It occurred to us that the explanation for these differences 
might be that there were different amounts of dibasic phosphates 
present in milk and that the acid converted the dibasic phosphate into 
acid phosphate, which increased the acidity but did not cause a posi- 
tive alcohol test. In order to test this theory one experiment was 
performed, the results of which are shown in Table 6. Two flasks of 
milk were used, each containing 50 c. c. of milk. One flask was left 
normal and 0.5 per cent dibasic sodium phosphate was added to the 
other. Various amounts of N/10 lactic acid were then added to each 
flask. As may be seen from the table, when 3 c. c. of N/10 lactic 
acid was added to the normal milk, the acidity was 2.37 and the 
alcohol test was positive with both 75 per cent and 68 per cent alco- 
hol. The flakes were large and medium, respectively. The same 
amount of acid added to the milk with dibasic phosphate increased 



THE ALCOHOL TEST IN RELATION TO MILK. 



11 



the acidity to 2.55 and yet the alcohol test was negative. When 7 
c. c. of N/10 lactic acid was added to the normal milk, the acidity was 
increased to 3.00 and the milk coagulated with large flakes with all 
the different percentages of alcohol. When 7 c. c. of N/10 lactic 
acid was added to milk with dibasic phosphate the acidity was 
increased to 3.05 and only the 75 per cent alcohol test was positive, 
and the coagulation was in the form of small flakes. When 8 c. c. 
of acid was added to the milk with dibasic phosphate the acidity 
was 3.19 and the alcohol test with both 75 per cent and 68 per cent 
.alcohol was positive. It was found by titration that 10 c. c. of a 
0.5 solution of dibasic phosphate required 1.56 c. c. of N/10 lactic 
acid to convert the dibasic into the monobasic phosphate; therefore 
50 c. c. of milk containing 0.5 per cent of dibasic sodium phosphate 
would require 7.8 c. c. of N/10 lactic acid to convert the dibasic 
into the monobasic phosphate. It will be seen from Table 6 that 
when from 7 to 8 c. c. of N/10 lactic acid was added to the milk 
with dibasic phosphate, the alcohol test became positive; that is, when 
the dibasic phosphate had been converted into monobasic phosphate 
then further increase in acidity caused a positive alcohol test. 

As a very general explanation of this result it may be said that when 
acid is added to milk it converts the dibasic phosphate into the 
monobasic phosphate. It follows that the acid and also the monobasic 
phosphate probably affect the casein and thereby change it into a 
condition in which it is possible to precipitate the casein by alcohol 
and cause a positive test. This action on the dibasic phosphate prob- 
ably explains in part the positive alcohol tests with different low 
acidities. 



Table 6. — Influence on the alcohol test of the addition of dibasic phosphate to milk. 







Normal milk. 




Normal milk + 0.5 per cent 


N/10 






dibasic sodium phosphate. 


lactic 
acid 




















added 




Alcohol test. 




Alcohol test. 


to 

50 c. c. 

of 


Acidity. 






Acidity. 




75 


68 


44 


75 


68 


44 


milk. 




per 


per 


per 




per 


per 


per 






cent. 


cent. 


cent. 




cent. 


cent. 


cent. 


c. c. 



1.S5 








2. 03 








1 


2.01 


t+S 


_ 


— 


2.13 


— 


— 


— 


3 


2.37 


+ L 


+M 


— 


2.55 


— 


— 


— 


5 


2.63 


+ L 


+ L 


+ S 


2.81 


— 


— 


— 


6 


2.80 


+ L 


+ L 


+ M 


2.92 


— 


— 


— 


7 


3.00 


+ L 


+ L 


+ L 


3.05 


+ S 


— 


— 


8 










3.19 


+ L 


+M 





9 










3.43 


+ L 


+ L 


_ 


11 










3.97 


+ L 


+ L 


+ L 











1 See footnote under Table 1. 



12 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



RESULT OF MIXING SOUR AND NORMAL MILK. 

Since a positive alcohol test may be produced by increasing the 
acidity, several investigators have pointed out that a mixture of sour 
and normal milks will give a positive test. The amount of sour milk 
which can be added to fresh milk without causing a positive alcohol 
test will, of course, depend upon the acidit}^ of the sour milk. In 
one experiment, the results of which are shown in Table 7, various 
percentages of sour, raw, and pasteurized milk were added to fresh 
milk. The addition of 1 per cent of sour milk caused a positive test 
with 75 per cent alcohol, 2.5 per cent caused a positive test with 68' 
per cent alcohol, and the addition of 10 per cent of sour milk was 
necessary to cause a positive test with 44 per cent alcohol. 

It must be taken into consideration in this experiment that the 
sour milk had a high acidity. If the acidity had been low a much 
higher per cent could undoubtedly have been added to the fresh 
milk without increasing the acidity sufficiently to cause a positive 
alcohol test. 

Table 7. — The alcohol test with a mixture of normal and sour milk. 



Addition of sour raw milk. Acidity 10.23. 


Addition of sour pasteurized milk. 
Acidity 9.87. 


Per 

cent of 
sour 
milk 

added. 


Acid- 
ity. 


Alcohol test. 


Per 

cent of 
sour 
milk 

added. 


Acid- 
ity. 


Alcohol test. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


0.0 
1.0 
2.5 
5.0 
10.0 


1.84 
1.93 
2.06 
2.30 
2.70 


»+s 

+M 
+ L 
+ L 


+M 
+ L 
+ L 


+M 






+s 

+ M 
+ L 
+ L 


+M 
+ L 
+ L 


+ VS 


1.0 
2.5 
5.0 
10.0 


1.88 
2.00 
2.29 
2.68 



1 See footnote under Table 1. 



In connection with the relation of acidity to the alcohol test the 
question arises as to whether or not the acidity of a sour milk can be 
neutralized so that the alcohol test will be negative. Some investi- 
gators have shown that the neutralization of the acidity does not 
cause a positive test to become negative, although the size of the 
flakes in the coagulation is somewhat reduced. We have tried one 
experiment in which various amounts of normal lactic acid were 
added to fresh milk, after which the acidity was reduced to the original 
acidity by the addition of sodium hydrate. From the results which 
are shown in Table 8 it will be seen that when the acidity was in- 
creased to 4.3, then neutralized to 1.90, the 68 per cent alcohol test 
was positive. The positive alcohol tests with 68 per cent alcohol 
could be made negative at acidities below 4.30 by reducing to about 
the original acidity of the normal milk. 



THE ALCOHOL TEST IN EELATION TO MILK. 13 

Table 8. — Effect on the alcohol test produced by neutralizing the acidity of milk. 



Amount 




Alcohol test. 




Alcohol test. • 


of normal 
lactic 










Acidity 
















acid 


Acidity. 








after neu- 








added to 




75 per 


68 per 


44 per 


tralizing. 


75 per 


68 per 


44 per 


50 c. c. 




cent. 


cent. 


cent. 




cent. 


cent. 


cent. 


of milk. 


















c.c. 


















0.0 
0.1 


1.84 
2.08 


i +M 


+M 


— 










1.80 


_ 


_ 


_ 


0.3 


2.42 


+ M 


+M 


— 


1.81 


— 


_ 


_ 


0.5 


2.99 


+L 


+M 


+VS 


1.64 


— 


— 


— 


0.8 


3.54 


+ L 


+L 


+M 


1.60 


+VS 


_ 


_ 


1.0 


3.94 


+ L 


+ L 


+L 


1.60 


+s 


_ 


— 


1.5 


4.30 


+ L 


+ L 


+L 


1.90 


+ L 


+M 


— 



1 See footnote under Table 1. 
EFFECT OF HEAT COMBINED WITH ACIDITY. 

As a matter of general interest we may mention the effect of heat- 
ing milk which gave a positive alcohol test. Auzinger (2) found 
that a milk which gave a positive test at 15° C. sometimes did not 
give the test when heated for 30 minutes at 60° C. Then again he 
found that the test might remain positive in milk heated to boiling. 
In Table 9 is shown the result of an experiment showing the effect 
of heat on the alcohol test with milk of two different acidities. No 
effect of heat was found on the sample of milk with an acidity of 
2.30, but there was a marked effect when the acidity was lower. 

We have no explanation to offer for this negative result of the test 
when the acidity is low. This action of heat might be of importance 
when the alcohol test is applied to pasteurized milk. 

Table 9. — Effect of heat on the alcohol test which is positive on account of acid action. 



Milk heated 
to— 


Alcohol test. 


Acidity, 2. 


Acidity, 2.30. 


75 per 
cent. 


68 per 
cent. 


75 per 
cent. 


68 per 
cent. 


°C. 
Not heated... 
40 .-. 


i+M 

+ M 
+ M 
+M 

+s 


+s 
+s 
+s 

+VS 


+L 
+ L 
+ L 
+ L 
+ L 
+ L 
+ L 


+ L 
+ L 
+ L 
+ L 
+ L 
+ L 
+ L 


60 


70 


80 


90 


100 





1 See footnote under Table 1. 



14 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



INFLUENCE OF THE ACTION OF RENNET. 

The relation of the alcohol test to the acidity of milk shows that 
acidity is one factor which may cause a positive alcohol reaction, 
but from the work of other investigators it is evident that it is not 
the sole cause. Morres throughout his papers points out that the 
alcohol test may be caused by an acid fermentation or by a rennet 
fermentation or by a mixture of both fermentations. 

In order to determine the effect of rennet action in relation to the 
test, we first tried the effect of prepared rennet. Four flasks of fresh 
milk were used and to each a different percentage of rennet was 
added. The milk in each flask was tested by the alcohol test at 
intervals of one hour. It will be seen from Table 10 that four 
different percentages of rennet were used, ranging from 0.00005 per 
cent to 0.0015 per cent. The acidity of the milk increased during 
the four hours from 1.64 to 1.70; therefore the influence of acidity 
can be neglected, since it is only a slight change. 

The results show that the action of rennet in milk may produce 
changes which cause a positive alcohol test and that two main factors 
are of importance, namely, the amount of rennet and the length of 
time the rennet has to act. Undoubtedly a third factor must be 
taken into consideration; that is, the temperature at which the milk 
is held. In our experiments the milk was held at room temperature. 
These results confirm those obtained by other investigators and 
indicate that the action of the rennet-forming bacteria might cause 
a positive alcohol reaction. 

Table 10. — Influence of rennet on the alcohol test. 





Rennet 
added. 


Alcohol test. 


Hours. 


75 per 


68 per 


44 per 






cent. 


cent. 


cent. 




Per cent. 











.00005 


— 


— 


— 




.00025 


— 


— 


— 




.0005 


— 


— 


— 




.0015 


— 


— 


— 


1 


.00005 


— 


— 


— 




.00025 


— 


— 


— 




.0005 


— 


— 


— 




.0015 


i +M 


+ M 


— 


2 


.00005 


— 


— 


— 




.00025 


— 


— 


— 




.0005 


+ M 


+ M 


— 




.0015 


+ L 


+ L 


+ M 


3 


.00005 


— 


— 


— 




.00025 


+M 


+M 


— 




.0005 


+ M 


+ M 


— 




.0015 


+ L 


+ L 


+ M 


4 


.00005 


— 


— 


— 




.00025 


+ L 


+L 


L 




.0005 


+ L 


+ L 


+ VS 




.0015 


( 2 ) 


( 2 ) 


( 2 ) 



1 See footnote under Table 1. 
8 Milk curdled by rennet. 



THE ALCOHOL TEST IN RELATION TO MILK. 



15 



In order to show the effect of rennet of bacterial origin, the action 
of a pure culture of a rennet-forming organism was studied. Two 
flasks of sterile skim milk were inoculated with different amounts of a 
pure culture of a rennet-forming organism. These flasks were 
incubated at 37° C, and the bacterial increase was determined at 
definite intervals, together with the alcohol test. The results are 
shown in Table 11. From a study of the table it is evident that 
rennet-forming bacteria will cause a positive alcohol test, but there 
must be a large bacterial increase to produce rennet enough to cause 
a positive test. 

The acidity was also increased during the incubation, but we believe 
this acidity played a minor part in causing the positive alcohol test. 

Table 11. — Influence on the alcohol test of rennet produced in milk by the growth of a pure 
culture of a rennet-forming organism. 



Experi- 
ment 
No. 


Age of 
cul- 
ture. 


Bacteria 

per cubic 

centimeter. 


Acid- 
ity. 


Alcohol test. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


I 
II 


Hours. 

2 
4 
5 
6 
7 

2 
4 


34,000 

62,000 

4,700,000 

9,000,000 

21,000,000 

31,000,000 

147,000 

200,000 

15,000,000 


1.98 

2.02 
2.06 
2.10 
2.11 
1.94 

2.10 


1 +L 
+L 

+ L 

+ L 


+ L 
+ L 

+ L 


+ L 

+s 



1 See footnote under Table 1. 



DIFFERENTIATION BETWEEN ACIDITY AND RENNET ACTION. 

The fact that reducing the acidity did not cause a negative alcohol 
test, as mentioned above, led us to believe that it might be possible 
to differentiate between a positive alcohol test caused by acidity and 
one caused by rennet action. In order to determine whether this 
was true two flasks of sterile skim milk were prepared. One was 
inoculated with a pure culture of a lactic-acid-forming organism and 
the other with equal amounts of a pure culture of lactic-acid bacteria 
and rennet-forming bacteria. The two flasks were then incubated at 
37° C. As may be seen from Table 12, the milk containing the 
lactic-acid bacteria had an acidity of 2.23 after 3 hours' incubation 
and the test was positive with both 75 per cent and 68 per cent 
alcohol. When the acidity was reduced to 1.49 all the alcohol tests 
were negative. The milk containing a mixed culture of lactic-acid 
bacteria and rennet-forming bacteria after 3 hours' incubation had 
an acidity of 2.32 and the alcohol test was positive with 75 per cent 
and 68 per cent alcohol. In both cases the coagulation was in the 
form of large flakes. When the acidity was reduced to 1.70 the 



16 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



alcohol test remained positive, although the size of the flakes was 
reduced. This milk after 5J hours' incubation had an acidity of 4.38 
and the milk coagulated with large flakes with each percentage of alco- 
hol. When the acidity was reduced to 1.49 the alcohol test remained 
positive, the only change being with the 44 per cent alcohol, in which 
case the size of the flakes was reduced. This experiment was re- 
peated, as will be seen from Table 12, and the results confirmed 
those of the first experiment. These results indicate that it may be 
possible to differentiate between an acid and an acid-and-rennet 
fermentation in milk, provided the acidity is not high. 

Table 12. — Differentiation between an acid and a mixed acid-and-rennet fermentation by 
neutralizing the acidity and using the alcohol test. 



Ex- 
peri- 
ment 

No. 


Pure culture 
of— 


After incubation at 37° C for 3 hours. 


After incubation at 37° 


C. for 5i hours. 


Acidity. 


Alcohol test. 


Acidity. 


Alcohol test. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


75 per 
cent. 


68 per 
cent. 


44 per 
cent. 


I. 


Lactic - acid 
bacteria. 

Mixtureof 
rennet- 
forming 
and lactic- 
acid bacte- 


2.23 

Neutralized to 
1.66. 

2.32 


>+L 
+L 


+L 
+L 


- 


3.21 

Neutralized to 
1.49. 

4.38 


+L 
+L 


+L 
+L 


+L 
+L 


II. 


ria. 

Lactic - acid 
bacteria. 


Neutralized to 
1.70. 


+S 


+VS 


- 


Neutralized to 
1.49. 

2 2.70 


+L 
+L 


+L 

+M 


+s 












M i x t u r e of 
rennet- 


3 2.00 


+L 


+M 


- 


a3.42 


+L 


+L 


+L 




forming 
and lactic- 
acid bacte- 




















ria. 


Neutralized to 
1.80. 


- 


- 


- 


Neutralized to 
1.60. 


+L 


+M 


+VS 



Note.— Acidity of normal milk in experiment 1, 1.75; in experiment II, 1.78. 

1 See footnote under Table 1. 

2 After 4i hours incubation at 37° C 

s Acidity after adding pure cultures to milk and before incubation. 



EFFECT OF HEAT COMBINED WITH RENNET ACTION. 

Earlier in this paper we have shown the effect of heat on the alcohol 
test with milk of high and low acidity, and as a matter of general 
interest the effect of heat on the alcohol test produced by rennet 
action may now be considered. The results of two experiments 
shown in Table 13 explain themselves clearly. Sufficient rennet was 
added to two samples of milk to cause a positive alcohol test with 75 
and 68 per cent alcohol. The milk was then heated, and it was found 
that at 90° C. the milk no longer gave a positive alcohol test. Both 
experiments showed the samo results. 



THE ALCOHOL TEST IN RELATION TO MILK. 



17 



Table 13. — Effect of heat on the alcohol test with milk in which the positive test is due to 

rennet action. 



Milk heated 
to— 


Alcohol test. 


75 per cent. 


6S per cent. 


Experi- 
ment I. 


Experi- 
ment II. 


Experi- 
ment I. 


Experi- 
ment II. 


°c. 

Not heated... 

40 

60 


>+L 
+L 

+ L 
+ L 
+ S 


+L 

+ L 
+L 

+L 
+VS 


+L 
+ L 
+L 
+L 

+s 


+ L 
+L 
+L 
+ L 
+VS 


70 

80 

90 



Note. 



-Acidity of milk in experiment I, 1.82; in experiment II, 1.84. 
1 See footnote under Table 1. 



The results which we have shown on the effect of rennet action in 
relation to the alcohol test confirm the work of other investigators, 
and it is evident that the rennet-forming group of bacteria in milk 
can play an important part in the production of a positive alcohol test. 

INFLUENCE ON THE ALCOHOL TEST OF CARBON DIOXTD IN MM. 

There are probably numerous minor factors which influence the 
alcohol test with market milk. While the two principal factors are 
probably acidity and the effect of rennet action, it is believed by some 
investigators that carbon dioxid plays a more or less important part. 
Auzinger (2) found that milk one hour old which gave a positive 
alcohol test gave a negative test after it had been held for 18 hours. 
He believes that carbon dioxid might be partly responsible for such a 
change. 

We have passed carbon-dioxid gas into milk many times and have 
always been able to cause a positive alcohol test. In one experiment 
carbon-dioxid gas was passed into milk until the acidity was 2.36 
(titration in cold milk with phenolphthalein as an indicator), and a 
positive alcohol test was obtained with 75 per cent and 68 per cent 
alcohol. As is shown in Table 14, this milk was heated at different 
temperatures up to 100° C. With the increase in temperature the 
acidity was reduced, due probably to the expelling of the C0 2 . Barille 
(4) has shown that carbon dioxid forms a very unstable compound, 
which he calls carbono phosphate of calcium and is easily broken up 
by heat. When the temperature reached 70° C, the alcohol test with 
68 per cent alcohol was negative and the acidity had been reduced 
from 2.36 to 2.05. At 90° C. the acidity was 1.91 and the alcohol 
test was negative with 75 per cent alcohol. 

There can be no doubt as to the fact that carbon dioxid may cause 
a positive alcohol test, provided there is a large enough amount in the 
82832°— Bull. 202—15—3 



18 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



milk. In order to determine how much carbon dioxid was required 
to cause a positive test with 68 per cent alcohol, the gas was passed 
into a flask of fresh milk until a positive alcohol test was produced. 
The amount of C0 2 in this milk and in the original milk was then 
determined. 1 It was found that the normal milk contained 0.76 per 
cent of C0 2 by volume at 32° C, and the milk through which the gas 
had been passed contained 13.05 per cent of C0 2 by volume. In this 
experiment it was necessary to increase the C0 2 content to 13.05 per 
cent by volume in order to cause a positive alcohol reaction with 
68 per cent alcohol. According to Kastle and Roberts (14) carbon 
dioxid is present in milk to the extent of 3 to 4 per cent by volume 
and partly escapes into the air when the milk is drawn. This being the 
case, it is evident that there is not enough carbon-dioxid gas in normal 
milk to cause of its own accord a positive alcohol test with 68 per cent 
alcohol. Of course, the presence of C0 2 may assist other factors to 
cause a positive alcohol test and in the case of bacterial fermentation 
where the gas is produced it might play a small part, but we believe 
that when 68 per cent alcohol is used in the test the influence of C0 2 
in mixed market milk would be very small, if it has any effect. 



Table 14.- 



-Effect of heat on alcohol test with milk-made acid to phenolphtlialein with 
carbon dioxid. 



Milk heated 
to— 


Acidity. 


Alcohol test. 


75 per cent. 


68 per cent. 


° C. 

Not heated 
40 
50 
60 
70 
80 
90 
100 

Original milk 


2.36 
2.32 
2.30 
2.19 
2.05 
2.05 
1.91 
1.92 
1.90 


+L 
+L 
+L 

+M 
+VS 


+L 
+ L 
+M 
+VS 



1 See footnote under Table 1. 



THE RELATION OF THE ALCOHOL TEST TO THE BACTERIA IN MIXED MARKET MILE. 

Having discussed the effect of acidity and the effect of rennet 
action on the alcohol test, let us consider the relation of the test to the 
bacteria in market milk. Since an increased acidity and also rennet 
action may cause a positive test, it is natural to suppose that there 
may be some definite relation between the alcohol test and the number 
of bacteria in milk, as the increase in the acidity and the rennet in 
milk is the result of bacterial growth. 

It is claimed by some authorities that the alcohol test is of great 
value for determining the freshness of milk, and as this is a question 

1 We are indebted to Dr. Clark, of the Dairy Division laboratory, for this analysis. 



THE ALCOHOL TEST IN RELATION TO MILK. 



19 



of great importance we have examined a number of samples of market 
milk which had been held for a number of days. 

In Table 1 5 are shown the results of the examination of four samples 
of market milk. One was raw milk and the others were pasteurized. 
Each bottle of milk was obtained from a different dairy and was held 
in a refrigerator at a temperature of about 9° C. The acidity and 
alcohol test were determined daily and bacterial counts were made on 
the first day and again when the 68 per cent alcohol gave a positive test. 

It is evident from the results obtained that the alcohol test (68 per 
cent) does not show the freshness of milk. The samples were held 
from 8 to 13 days at 9° C. before the alcohol test became positive, and 
during that time the bacteria had increased to more than 100,000,000 
per cubic centimeter. 

Table 15. — Effect on the alcohol test of holding milk at 9° C. 



Days 
held. 


Raw milk. 


Pasteurized milk A. 


Acid- 
ity. 


Alcohol test. 


Bacteria 

per cubic 

centimeter. 


Acid- 
ity. 


Alcohol test. 


Bacteria 
per cubic 
centimeter. 


75 
per 
cent. 


68 
per 
cent. 


44 
per 
cent. 


75 

per 
cent. 


68 
per 

cent. 


44 
per 
cent. 



1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 


1.72 


i+V.S. 

+v. s. 
+v. s. 
+v. s. 


- 


- 


8,200 




- 


- 


— 


37,000 




















1.88 
1.95 
2.02 


+v. s. 

+ V. S. 


— 


— 






+s. 
+s. 


- 


- 


















1.78 
1.88 
1.93 
















2.08 
2.43 
2.72 


+ M. 
+ L. 
+ L. 


+M. 
+ L. 


- 




2.30 

2.48 
2.75 


+L. 
+L. 

-Un- 


+M. 

+ L. 
+ L. 


+ L. 


242,000,000 




146,000,000 


626,000,000 


Days 
held. 


Pasteurized milk B. 




pasteurized 


milkC 


Acid- 
ity. 


Alcohol test. 


Bacteria 

per cubic 

centimeter. 


Acid- 
ity. 


Alcohol test. 


Bacteria 

per cubic 

centimeter. 


75 
per 
cent. 


68 
per 
cent. 


44 
per 
cent. 


75 
per 
cent. 


68 
per 
cent. 


44 
per 

cent. 



1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 


1.74 


+ M. 
+ L. 

+M. 
+M. 


= 


- 


182,000 




- 


- 




133,000 




1.75 














1.88 

2.00 
2.10 
2.38 


+M. 
+U. 
+M. 
+M. 

+M. 
+ L. 


+M. 
+M. 
+ L. 


— 






+M. 
+ L. 
+ L. 


+M. 
+ L. 
+ L. 


+M. 
















145,000,000 


2.12 
2.34 
2.77 


221,000,000 




650,000,000 




2.45 


+ L. 


+ L. 


+M. 


700,000,000 































































1 See footnote under Table 1. 



20 



BULLETIN 202, TJ. S. DEPARTMENT OF AGRICULTURE. 



EFFECT OF HOLDING THE MILK AT DIFFERENT TEMPERATURES. 

The temperature at which the milk is held would, of course, 
affect the length of time before the alcohol test becomes positive. 
To show the effect of temperature we held samples of raw and pas- 
teurized milk at 9° C. and also at room temperature, and examined 
them in the same manner as in the preceding experiment. The 
results are shown in Table 16. In order to have about the same 
bacterial content in the milk held at the different temperatures, a 
quart of milk was thoroughly mixed and placed in sterilized pint 
bottles. 

The results show clearly that the temperature at which milk is held 
has a marked influence on the time when the alcohol test will be posi- 
tive. Also, as shown by sample of raw milk C, the bacterial content 
of the milk is an important factor. In all the samples it will be no- 
ticed that the bacterial counts show an exceedingly high number 
when the 68 per cent alcohol test was positive. 



Table 16. — Comparison of the alcohol tests with milk held at 9° C. and at 24° C. 



Temper- 
ature at 
which 
held. 


Days 
held. 


Raw milk A. 


Pasteurized milk B. 


Acid- 
ity. 


Alcohol test. 


Bacteria per 
cubic centi- 
meter. 


Acid- 
ity. 


Alcohol test. 


Bacteria per 
cubic centi- 
meter. 


75 
per 
cent. 


68 
per 
cent. 


44 
per 
cent. 


75 
per 
cent. 


68 
per 
cent. 


44 
per 
cent. 


9° C. . . 

Room 
tem- 
pera- 
ture 12 




1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

17 



l(9a.m.) 
l(3p.m.) 


1.85 
1.85 
1.90 
1.80 
1.79 


= 


- 


- 


1,600 


1.65 
1.60 
1.60 
1.70 
1.71 


= 


- 


- 


139,000 


















1.85 
1.90 
1.80 

1.80 


= 


- 


= 




1.60 
1.80 
1.65 


H-S 


— 


~ 


















1.72 
2.00 






197,000,000 




2.70 
4.1 

1.85 
1.90 
3.1 


+L 
+ L 

+ L 


+ M 
+ L 

+ L 


+L 

+ L 


400,000,000 


2.3 


+ L 


+M 


- 


960,000,000 


440,000,000 

2,900 
"534,666,000 


4.3 

1.65 
1.85 
3.70 


+L 

+ S 
+ L 


+ L 

+ L 


+L 

+ L 




146,000 
32.600,000 
694,000,000 



1 See footnote under Table 1. 



a About 24° C. 



THE ALCOHOL TEST IN RELATION TO MILK. 



21 



Table 16. — Comparison of the alcohol tests with milk held at 9° C. and at 24° C. — 

Continued. 



Temper- 
ature at 
which 
held. 


Days, 
held. 


Raw milk C. 


Acid- 
ity. 


Alcohol test. 


Bacteria per 
cubic centi- 
meter. 


75 
per 
cent. 


68 
per 
cent. 


44 
per 
cent. 


9°C 

Room 
tem- 
pera- 
ture' . 



1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
17 


l(9a.m.) 


1.85 

1.80 
1.95 
2.35 
2.36 


+ M 
+ L 


+s 

+L 


[ 


7,870,000 


93,000,000 
130,000,000 
188,000,000 


2.88 


+ L 


+L 


+M 


430,000,000 


















































































1.85 
4.90 


_ 


_ 





7,610,000 
Curdled. 









1 See footnote under Table 1. 



2 About 24° C. 



At various times different investigators have used the alcohol test 
on market milk. Aurnhammer (1) in an examination of 250 samples 
of market milk during July and August of 1907 found the 68 per cent 
alcohol test positive in 82 samples. In a study of market milk in 
Philadelphia, Campbell (5) found that 37 of 100 samples of milk gave 
a positive test with 68 per cent alcohol. Of these 37 samples 17 
contained less than and 20 more than 1,000,000 bacteria per cubic 
centimeter. It was found by Nurenberg and Lythgoe (23) during an 
examination of 2,600 samples of market milk that only 63 gave a 
positive test with 68 per cent alcohol. 

We made alcohol tests on 236 samples of Washington market 
milk during the period from March 20 to June 4, 1914. These 
samples and their bacterial counts were supplied by the Health 
Department, District of Columbia. 1 Of the 236 samples we found 
that 37 gave an alcohol test with 75 per cent alcohol, 20 with 68 per 
cent alcohol, and 5 with 44 per cent alcohol. The samples which 
gave a positive test are tabulated in Table 17 with their acidity and 
bacterial counts. There were 177 samples of raw milk and 59 samples 
of pasteurized milk in the 236 samples examined. As may be seen 
from the table, 35 of the raw-milk samples gave a positive test with 
75 per cent alcohol and only 2 of the 59 samples of pasteurized milk. 

i We take this occasion to express our thanks to Dr. Kinyoun and Dr. Dieter, of the Health Depart- 
ment, for the samples of market milk and their bacterial counts which they so kindly furnished us. 



22 BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 

Table 17. — Raw and pasteurized market milk which gave positive alcohol tests. 





Sam- 
ple 






Alcohol tests. 


Milk. 


Acidity. 


Bacteria per 
cubic 
















ber. 




centimeter. 


75 


68 


44 








per cent. 


per cent. 


per cent. 




1 


2.01 


2.100 


i + M 


+VS 






2 


2.30 


7,000 


+ L 


+M 


_ 




3 


2.00 


12,000 


+ M 


— 


— 




4 


1.60 


14,000 


+M 


+s 


— 




5 


1.97 


18,000 


+M 


— 


— 




6 


1.92 


24,000 


+M 


+s 


+VS 




7 


1.91 


24,000 


+ S 


_ 


— 




8 


1.70 


29, 000 


+S 


— 


— 




9 


1.62 


51,000 


+M 


+s 


_ 




10 


2.30 


67, 000 


+ L 


+M 


_ 




11 


1.79 


121,000 


+M 


+ S 


+ VS 




12 


1.75 


156,000 


+s 


— 


— 




13 


2.00 


200,000 


+M 


— 


— 




14 


1.94 


350,000 


+ M 


— 


_ 




15 


1.90 


442,000 


+s 


— 


— 




16 


2.06 


464,000 


+L 


+M 


— 




17 


1.95 


1,200,000 


+M 


+VS 


— 




18 


1.76 


1,300,000 


+ VS 


— 


— 




19 


1.75 


1,500,000 


+s 


— 


— 




20 


1.80 


1,600,000 


+M 


+s 


— 




21 


1.70 


2, 100, 000 


+M 


— 


— 




22 


2.03 


2,120,000 


+ S 


— 


— 




23 


1.90 


2,200,000 


+S 


— 


— 




24 


1.95 


2,300,000 


+M 


+s 


— 




25 


2.45 


2,600,000 


+ L 


+M 


+VS 




26 


1.95 


4, 100, 000 


+ M 


+M 


— 




27 


1.93 


4,700,000 


+VS 


— 


— 




28 


2.15 


7,200,000 


+M 


+ S 


— 




29 


2.19 


8,600,000 


+M 


+s 


— 




30 


3.65 


10,200,000 


+L 


+ L 


+ L 




31 


1.90 


10,500,000 


+ M 


— 


— 




32 


2.05 


20,200,000 


+M 


— 


— 




33 


1.95 


20, 400, 000 


+M 


+M 


— 




34 


2.55 


20,600,000 


+ L 


+ L 


+ L 




35 


2.10 


21,200,000 


+ L 


+M 


— 


Pasteurized.. 


1 


1.68 


2,000 


+8 


— 


— 




2 


1.90 


8,000 


+M 


+ S 


— 



1 See footnote under Table 1. 
RESULTS OF TESTS WITH SAMPLES OF KNOWN BACTERIAL CONTENT. 

When we consider the alcohol test in relation to the number of 
bacteria in milk, a short survey of the results is sufficient to show 
that there is no definite relation. Of the 35 samples of raw milk 
which showed a positive test with 75 per cent alcohol, 16, or 45.7 
per cent, contained less than 500,000 bacteria per cubic centimeter. 
Of the 19 samples positive with 68 per cent alcohol, 8, or 42.1 per cent, 
contained less, and 11, or 57.9 per cent, more than 500,000 bacteria 
per cubic centimeter. Of the 5 samples positive with 44 per cent 
alcohol, 2 samples, or 40 per cent, contained less, and 3, or 60 per cent, 
more than 500,000 bacteria per cubic centimeter. The number of 
bacteria in samples which gave a positive alcohol test ranged from 
2,100 to 21,200,000 per cubic centimeter. 

The samples of pasteurized milk which showed a positive alcohol 
test had a very low bacterial count. 

In order further to show that the alcohol test has no definite 
relation to the bacterial count, there are tabulated in Tables 18 and 19 
the samples of raw and pasteurized milk which gave negative alcohol 



THE ALCOHOL TEST IN RELATION TO MILK. 



23 



tests, together with their acidity and bacterial counts. We wish to 
call particular attention to the bacterial counts of 142 samples of 
raw milk which ranged from 2,000 to 19,600,000 bacteria per cubic 
centimeter. Of these 142 samples none gave a positive alcohol test, 
yet 86, or 60.6 per cent, contained less than and 39.4 per cent more 
than 500,000 bacteria per cubic centimeter. 

The bacterial counts of the samples of pasteurized milk which gave 
a negative alcohol test ranged from 1,200 to 3,600,000 per cubic 
centimeter, as may be seen from Table 19. 

From our results we believe that there is no definite relation between 
the alcohol test and the bacterial count, except in special cases where 
the bacteria have developed to a point where there is sufficient acid 
produced or where rennet-forming bacteria have acted sufficiently 
to influence the test. 



Table 18.— Acidity and bacterial count of samples of raw market milk which gave negative 
alcohol tests with 75 per cent, 68 per cent, and 44 per cent alcohol. 



Sam- 


Acid- 
ity. 


Bacteria 


Sam- 


Acid- 
ity. 


Bacteria 


Sam- 


Acid- 
ity. 


Bacteria 


ple 


per cubic 


ple 


per cubic 


ple 


per cubic 


No. 


centimeter. 


No. 


centimeter. 


No. 


centimeter. 


1 


1.08 


2,000 


49 


1.82 


82,000 


97 


1.95 


812,000 


2 


2.10 


5,000 


50 


1.75 


86,000 


98 


1.85 


840, 000 


3 


1.85 


6,000 


51 


1.08 


92,000 


99 


1.80 


860,000 


4 


1.75 


7,000 


52 


1.90 


93,000 


100 


1.60 


880,000 


5 


1.82 


8,000 


53 


1.85 


93,000 


101 


1.62 


906,000 


6 


2.05 


8,000 


54 


1.60 


105,000 


102 


1.69 


910,000 


7 


2.05 


8,500 


55 


1.70 


109,000 


103 


1.90 


910,000 


8 


1.83 


11,000 


56 


1.93 


115,000 


104 


1.90 


910,000 


9 


1.92 


13,000 


57 


2.00 


118,000 


105 


1.80 


920,000 


10 


1.97 


13,000 


58 


1.87 


120,000 


106 


1.75 


1,040,000 


11 


1.75 


14,000 


59 


2.06 


120,000 


107 


1.75 


1,100,000 


12 


2.00 


14,000 


60 


1.75 


130,000 


108 


1.75 


1,170,000 


13 


2.15 


16,000 


61 


1.75 


132,000 


109 


1.65 


1,200,000 


14 


2.10 


18,000 


62 


2.00 


147,000 


110 


1.70 


1,200,000 


15 


1.75 


21,000 


63 


1.85 


149,000 


111 


1.90 


1,210,000 


16 


1.95 


21,000 


64 


1.90 


150,000 


112 


1.70 


1,400,000 


17 


1.62 


22,000 


65 


1.90 


157, 000 


113 


1.85 


1,400,000 


18 


2.15 


22,000 


66 


1.48 


160,000 


114 


1.85 


1,460,000 


19 


1.80 


25,000 


67 


2.00 


164,000 


115 


1.80 


1,600,000 


20 


1.52 


26,000 


68 


1.85 


172,000 


116 


2.08 


1,600,000 


21 


1.80 


26,000 


69 


1.80 


206,000 


117 


1.70 


1,630,000 


22 


1.95 


26,000 


70 


2.00 


210,000 


118 


1.98 


1,710,000 


23 


1.89 


27,000 


71 


2.03 


212,000 


119 


2.20 


1,800,000 


24 


1.65 


29,000 


72 


1.90 


214,000 


120 


2.10 


2, 120, 000 


25 


2.00 


32,000 


73 


1.85 


216,000 


121 


1.74 


2, 210, 000 


26 


1.60 


33,000 


74 


2.00 


220,000 


122 


1.75 


2, 260, 000 


27 


1.84 


33,000 


75 


1.82 


238,000 


123 


1.85 


2,340,000 


28 


1.65 


34,000 


76 


1.83 


238,000 


124 


1.90 


2,580,000 


29 


1.76 


34,000 


77 


2.00 


242,000 


125 


1.85 


2,710,000 


30 


2.10 


35,000 


78 


1.70 


266,000 


126 


1.71 


2,840,000 


31 


1.90 


36,000 


79 


2.02 


268,000 


127 


1.70 


2,900,000 


32 


1.97 


36,000 


80 


1.90 


270,000 


128 


1.80 


2,920,000 


33 


1.74 


37,000 


81 


1.80 


278,000 


129 


1.70 


3,300,000 


34 


1.75 


37,000 


82 


1.80 


310,000 


130 


2.07 


3,800,000 


35 


1.55 


38,000 


83 


1.80 


350,000 


131 


1.85 


4,300,000 


36 


1.80 


38; 000 


84 


1.75 


360,000 


132 


1.96 


4,800,000 


37 


2.00 


39,000 


85 




422,000 


133 


2.15 


5,100,000 


38 


1.75 


42,000 


86 


2.10 


451,000 


134 


2.05 


5,300,000 


39 


1.81 


42,000 


87 


1.80 


506,000 


135 


1.80 


5,700,000 


40 


1.70 


43,000 


88 


2.05 


510,000 


136 


1.80 


6,400,000 


41 


1.80 


46,000 


89 


1.90 


560,000 


137 


1.85 


6,900,000 


42 


1.78 


51,000 


90 


1.75 


610,000 


138 


1.90 


6,900,000 


43 


1.90 


54,000 


91 


2.10 


620,000 


139 


1.80 


8,800,000 


44 


1.86 


56,000 


92 


1.88 


624,000 


140 


1.90 


12,600,000 


45 


1.60 


63,000 


93 


1.70 


640, 000 


141 


1.85 


12, 700, 000 


46 


1.75 


69,000 


94 


1.74 


740,000 


142 


2.15 


19,600,000 


47 


1.90 


74,000 


95 


1.75 


740,000 








48 


1.71 


79,000 


96 


2.00 


800,000 









24 



BULLETIN 202, U. S. DEPARTMENT OP AGRICULTURE. 



Table 19. — Acidity and bacterial count of samples of pasteurized market milk which 
gave negative alcohol tests with 75 per cent, 68 per cent, and 44 per cent alcohol. 



Sam- 


Acid- 
ity. 


Bacteria 


Sam- 


Acid- 
ity. 


Bacteria 


Sam- 


Acid- 
ity. 


Bacteria 


ple 
No. 


per cubic 


ple 


per cubic 


ple 


per cubic 


centimeter. 


No. 


centimeter. 


No. 


centimeter. 


1 


1.85 


1,200 


20 


1.75 


15,000 


39 


1.85 


104,000 


2 


1.77 


1,200 


21 


1.70 


16,000 


40 


2.05 


110,000 


3 


1.66 


1,900 


22 


1.65 


16,000 


41 


1.65 


114,000 


4 


1.75 


3,000 


23 


2.05 


16,000 


42 


1.76 


120,000 


5 


1.66 


4,000 


24 


1.69 


17,500 


43 


1.80 


133,000 


6 


1.80 


5,000 


25 


1.78 


21,000 


44 


1.75 


194,000 


7 


1.85 


7,000 


26 


1.66 


21,000 


46 


1.73 


264,000 


8 


1.85 


7,600 


27 


1.80 


24,000 


47 


1.70 


284,000 


9 


1.80 


8,000 


28 


1.83 


32,000 


48 


1.90 


340,000 


10 


1.80 


9,000 


29 


1.67 


37,000 


49 


1.76 


446,000 


11 


1.71 


9,000 


30 


1.96 


41,000 


50 


1.85 


720,000 


12 


1.75 


11,000 


31 


1.75 


52,000 


51 


1.65 


740,000 


13 


1.85 


11,000 


32 


1.90 


59,000 


52 


1.75 


940,000 


14 


1.70 


11,000 


33 


1.85 


62, 000 


53 


1.74 


1,280,000 


15 


1.65 


12,000 


34 


1.85 


64,000 


54 


1.60 


1,660,000 


16 


1.85 


13,000 


35 


1.70 


65,000 


55 


1.97 


2,460,000 


17 


1.75 


14,000 


36 


1.75 


68,000 


56 


1.60 


3,100,000 


18 


1.75 


15,000 


37 


1.80 


71,000 


57 


2.00 


3,600,000 


19 


1.80 


15,000 


38 


1.70 


74,000 









In the early stages of the growth of acid-forming bacteria in milk, 
when the numbers are low, there is a period in which a rapid increase 
in numbers takes place without any increase in acidity which can be 
detected by ordinary chemical methods, or it may occur with only a 
slight increase in acidity ; consequently if the alcohol test were made 
during that period there would be a high bacterial count and yet not 
high acidity enough to cause a positive alcohol test. The same is 
true of the action of the rennet-forming bacteria in their growth and 
action, as we have shown earlier in this paper when dealing with the 
relation of acidity, and also the effect of rennet on the alcohol test. 
Besides these facts there are other groups of bacteria which may 
develop in milk and yet have no influence on the alcohol test, as, for 
example, the alkali-forming group of bacteria. We have tried cul- 
tures of this group of organisms and found that they did not produce 
a positive alcohol test. There are other groups of bacteria in the 
flora of milk, such as the inert group, which also would probably 
develop without influencing the alcohol test in any way. When we 
consider all these facts it is not strange that there is no definite 
relation between the bacterial flora of milk and the bacterial count. 

When the 68 per cent alcohol test is positive with a sample of 
market milk, it is evidence that there is some change in the milk from 
normal. In some cases it may be due to an increased acidity and in 
consequence a change in the casein of the milk, due to bacterial 
action. In other cases it may be due to a pure rennet fermentation 
or there may be a combination of an acid-and-rennet fermentation. 
In such cases the bacterial count would undoubtedly be high. How- 
ever, there still remains to be explained the reason for a positive 
alcohol test in samples of market milk with a low bacterial count and 
low acidity. 



THE ALCOHOL TEST IN RELATION TO MILK. 25 

We can not see that the alcohol test is of any particular value in 
the control of a market milk supply except as a means of evidence 
that milk from a particular source is abnormal in some way and 
should be examined by other tests. It might be of value at a re- 
ceiving station as a means of detecting sour milk, but the test would 
be expensive compared with the use of alkaline tablets for the rapid 
determination of acidity as described by Farrington and Woll (9). 

THE TITRATION METHOD OF APPLYING THE ALCOHOL TEST. 

From the simple alcohol test in which a definite volume of a definite- 
percentage alcohol is added to an equal volume of milk there has 
developed a method in which a definite volume of milk is titrated 
with certain percentages of alcohol until a coagulation of the milk is 
produced. 

Lohnis (16) has found this titration method to be of value as a test 
for the quality of market milk. He found that there was quite a 
definite relation between the titration with 80 per cent alcohol and 
the bacterial content of market milk. He titrated 2 c. c. of milk in a 
beaker against a black background with 90, 80, and 70 per cent alco- 
hols, the titration being made at a temperature of from 15° to 20° C. 
The first appearance of flakes was considered the end point. 

We have used this method in the titration of 116 samples of market 
milk furnished with bacterial counts by Dr. Kinyoun and Dr. Dieter, 
of the Health Department of the District of Columbia. In our 
titrations of 92 samples of raw and 24 samples of pasteurized milk we 
have not found any definite relation between the titration with 90 
per cent and 80 per cent alcohols and the bacterial count. In Table 
20 is shown the acidity, bacterial counts, and alcohol titration of 92 
samples of raw milk, and in Table 21 the results of an examination 
of 24 samples of pasteurized milk. The bacterial counts of the raw 
milk ranged from 2,100 to 20,600,000 per cubic centimeter, and the 
pasteurized milk from 1,200 to 3,100,000 bacteria per cubic centimeter. 
Consequently we were able to titrate samples having a great variation 
in their bacterial content. If a study is made of the bacterial counts 
and the alcohol titrations shown in Tables 20 and 21 it will be seen 
that there is no definite relation between them. In order to bring 
this point out more clearly the titrations of samples containing more 
than 500,000 and less than 500,000 bacteria per cubic centimeter 
have been averaged, as shown in Table 22. The average titration 
with 90 per cent alcohol of 46 samples of raw milk containing more 
than 500,000 bacteria per cubic centimeter was 1.95 c. c, while the 
average titration of 46 samples containing less than 500,000 per cubic 
centimeter was 2.39 c. c. The average titration of 46 samples with 
80 per cent alcohol was 4.61 c. c. when the bacterial count was more 



26 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



than 500,000 per cubic centimeter and 5.61 c. c. when the counts 
were less than 500,000 per cubic centimeter. The average titrations 
of the pasteurized milk samples showed even smaller differences. 
The small differences in the average titration of samples with a high 
and a low bacterial count show that there is little, if any, relation 
between the alcohol titration and the bacterial count. This is shown 
even more strikingly in Table 23, where the range in titrations 
among samples grouped according to bacterial counts is recorded. 
With these extreme ranges among samples of milk with high and low 
bacterial contents it would be almost impossible to interpret an 
alcohol titration in terms of bacteria. 

Table 20. — Alcohol titrations of raw market milk. 









Alcohol titra- 








Alcohol titra- 


Sam- 
ple 
No. 




Bacteria 


tion. 


Sam- 
ple 
No. 




Bacteria 


tion. 


Acid- 
ity. 


per cubic 
centi- 






Acid- 
ity. 


per cubic 
centi- 
















meter. 


90 per 


80 per 




meter. 


90 per 


80 per 








cent. 


cent. 








cent. 


cent. 








c. c. 


c. c. 








c. c. 


c. c. 


1 


2.01 


2,100 


1.03 


1.86 


47 


1.80 


506,000 


1.50 


1.80 


2 


2.30 


7,000 


.51 


.93 


48 


1.75 


610,000 


2.93 


5.25 


3 


1.75 


7,000 


2.17 


5.30 


49 


2.10 


620,000 


1.80 


5.10 


4 


1.82 


8,000 


2.76 


4.54 


50 


1.88 


624, 000 


2.91 


9.20 


5 


2.05 


8,500 


2.00 


5.30 


51 


1.74 


740, 000 


2.96 


6.80 


6 


1.75 


14,000 


2.21 


4.67 


52 


1.75 


812, 000 


1.91 


6.76 


7 


2.15 


16,000 


2.80 


7.60 


53 


1.80 


860,000 


1.62 


3.00 


8 


1.95 


21,000 


2.70 


7.43 


54 


1.02 


906,000 


1.68 


3.30 


9 


1.91 


24,000 


1.59 


3.08 


55 


1.69 


910,000 


2.08 


6.13 


10 


1.80 


25,000 


3.29 


11.73 


56 


1.80 


920,000 


1.85 


4.50 


11 


1.52 


26,000 


3.12 


7.28 


57 


1.75 


1,040,000 


1.92 


4.37 


12 


1.65 


29,000 


2.68 


10.07 


58 


1.75 


1.100,000 


2.36 


4.00 


13 


2.00 


32,000 


3.64 


9.59 


59 


1.75 


1,170,000 


2.20 


6.04 


14 


1.60 


33,000 


2.56 


5.55 


60 


1.65 


1,200,000 


1.90 


5.76 


15 


1.65 


34,000 


2.48 


5.51 


61 


1.76 


1,300,000 


2.72 


6.59 


16 


2.10 


35,000 


1.24 


2.71 


62 


1.70 


1,400,000 


1.63 


3.74 


17 


1.75 


37,000 


2.26 


4.40 


63 


1.85 


1,400,000 


2.36 


4.42 


18 


1.80 


38,000 


2.71 


6.50 


64 


1.85 


1,400,000 


3.04 


9.61 


19 


1.55 


38,000 


3.06 


6.92 


65 


1.80 


1,600,000 


1.23 


5.40 


20 


1.75 


42,000 


3.00 


4.28 


66 


2.08 


1,600,000 


2.51 


5.94 


21 


1.80 


46,000 


2.78 


9.68 


67 


1.70 


1,630,000 


2.40 


6.00 


22 


2.30 


67,000 


.50 


1.1 


68 


2.20 


1,800,000 


1.30 


2.37 


23 


1.75 


69,000 


2.58 


5.98 


69 


1.70 


2,100,000 


1.16 


1.60 


24 


1.90 


74,000 


3.03 


6.33 


70 


2.03 


2, 120, 000 


1.16 


2.34 


25 


1.71 


79,000 


3.50 


6.91 


71 


1.74 


2,210,000 


2.10 


3.96 


26 


1.85 


93,000 


1.57 


4.36 


72 


1.75 


2,260,000 


1.81 


4.12 


27 


1.70 


109,000 


3.04 


5.20 


73 


1.85 


2,340,000 


2.42 


4.78 


28 


1.87 


120,000 


2.46 


5.80 


74 


2.45 


2,600,000 


.78 


.97 


29 


1.75 


130,000 


1.60 


3.20 


75 


1.71 


2,840,000 


2.95 


3.97 


30 


1.75 


132,000 


2.72 


10.45 


76 


1.70 


2, 900, 000 


2.12 


6.36 


31 


2.00 


147, 000 


1.72 


3.80 


77 


1.80 


2, 920, 000 


3.00 


6.85 


32 


1.85 


149,000 


3.54 


8.17 


78 


1.70 


3,300,000 


2.80 


6.30 


33 


1.80 


160,000 


1.48 


2.49 


79 


2.00 


3,600,000 


1.90 


5.40 


34 


1.80 


206, 000 


3.16 


5.52 


80 


1.85 


4,300,000 


2.05 


5.00 


35 


2.00 


210, 000 


2.68 


7.00 


81 


1.93 


4,700,000 


1.40 


3.90 


36 


2.03 


212,000 


2.35 


5.70 


82 


1.96 


4,800,000 


1.46 


2.70 


37 


1.90 


214,000 


1.68 


4.52 


83 


2.05 


5,300,000 


1.60 


2.68 


38 


1.82 


238,000 


2.75 


7.00 


84 


1.80 


5,700,000 


2.94 


7.25 


39 


2.00 


242,000 


1.24 


2.90 


85 


1.85 


6,900,000 


1.01 


2.41 


40 


1.76 


266,000 


1.95 


3.94 


86 


2.19 


8, 600, 000 


.92 


1.86 


41 


2.02 


268,000 


2.57 


5.20 


87 


1.80 


8,800,000 


2.96 


10.83 


42 


1.90 


270,000 


2.74 


4.45 


88 


1.90 


12,000,000 


1.83 


5.10 


43 


1.80 


278,000 


3.22 


8.70 


89 


1.85 


12.700,000 


1.40 


2.43 


44 


1.80 


310,000 


2.34 


4.58 


90 


2.15 


19,600,000 


1.56 


3.10 


45 


1.80 


350, 000 


2.68 


4.67 


91 


1.95 


20,400,000 


.80 


1. 40 


46 


1.75 


360,000 


2.35 


5.29 


92 


2.55 


20,000,000 


.52 


80 



THE ALCOHOL TEST IN RELATION TO MILK. 27 

Table 21. — Alcohol titrations of pasteurized market milk. 









Alcohol titra- 








Alcohol titra- 


Sam- 




Bacteria 


tion. 


Sam- 




Bacteria 


tion. 


ple 
No. 


Acid- 
ity. 


per cubic 
centimeter. 




ple 
No. 


Acid- 
ity. 


per cubic 
centimeter. 




















90 per 


80 per 








90 per 


80 per 








cent. 


cent. 








cent. 


cent. 








c. c. 


c. c. 








c. c. 


c. c. 


1 


1.77 


1,200 


2.20 


5.74 


13 


1.70 


65,000 


2.88 


5.04 


2 


1.70 


11,000 


2.84 


6.76 


14 


1.75 


68,000 


2.40 


5.50 


3 


1.65 


12,000 


3.08 


8.80 


15 


1.80 


71,000 


2.50 


4.49 


4 


1.85 


13,000 


1.56 


4.43 


16 


1.85 


104,000 


2.43 


3.96 


5 


1.75 


14,000 


1.65 


3.10 


17 


1.70 


120, 000 


2.10 


5.96 


6 


1.75 


15,000 


2.22 


3.42 


18 


1.75 


194, 000 


3.05 


6.01 


7 


1.70 


16,000 


2.76 


4.10 


19 


1.73 


264, 000 


2.24 


3.00 


8 


1.78 


21,000 


2.33 


5.91 


20 


1.70 


284,000 


2.86 


5.48 


9 


1.80 


24,000 


1.69 


6.10 


21 


1.76 


446, 000 


1.82 


5.34 


10 


1.90 


59,000 


2.57 


3.68 


22 


1.60 


1,600,000 


2.10 


4.78 


11 


1.85 


62,000 


2.23 


5.47 


23 


1.97 


2,460,000 


2.08 


3.66 


12 


1.60 


63,000 


2.53 


3.24 


24 


1.60 


3,100,000 


3.53 


7.07 



Table 22. — Average alcohol titrations of samples of raw and pasteurized market milk in 

tables 20 and 21. 



Milk. 


Number 

of 
samples. 


Bacteria per cubic 
centimeter. 


Average alcohol 
titration. 


90 per 
cent. 


80 per 
cent. 




46 

46 

3 

21 


More than 500,000 . 
Less than 500,000. . 
More than 500,000.. 
Less than 500,000. . 


c. c. 
1.95 
2.39 
2.57 
2.28 


c. c. 
4.61 
5.61 
5.17 
5.02 


Pasteurized . . 



Table 23. — Range in alcohol titrations of market milk shown in detail in tables 20 and 21. 



Bacteria per cubic centimeter. 


Alcohol titration. 


90 per cent. 


80 per cent. 


Lowest. 


Highest. 


Lowest. 


Highest. 


26 samples with less than 100,000 

30 samples with from 100,000 to 1,000,000 
36 samples with over 1 ,000,000 


c. c. 
0.51 
1.24 
.52 


c. c. 
3.64 
3.54 
3.04 


c. c. 
0.93 
1.80 
.60 


c. c. 
11.73 
10.45 
10.83 





For the sake of clearness we have plotted in figure 1 the bacterial 
counts and the 90 per cent alcohol titration. In this figure the 
titrations of 116 samples of milk were plotted as ordinates and the 
logarithms of the bacterial counts as abscissae. The numbers 3, 4, 
5, 6, 7, and 8 represent the mantissa of the logarithms of the bacterial 
counts. Consequently from 3 to 4 was plotted the logarithm of 
samples with a bacterial count of from 1,000 to 9,999, from 4 to 5 
counts from 10,000 to 99,999, and so on, as may be seen from the 
figure. By this method of plotting it is possible to plot bacterial 
counts ranging from low to high numbers, which would otherwise be 



28 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



impossible in a limited space. A glance at the plot which shows the 
90 per cent alcohol titration and the bacterial count of 116 samples 
indicates clearly that there is no definite relation between them. In 
figure 2 we have plotted in the same way the 80 per cent alcohol 



Si 
IS 

ft o 



! — r~ i ""**r"^ , ~" 1- r~ i ^ " rt — 7* *"• — ' — " 

| • , • i \ i . — f »*n — t - * • 

„ i 1_j '-& L . . J— _, 

\> J» s & 7 <9 



V 



1-9,999 «4* 10,000-93,999 «4* 100,000-999,999 ^*fOOO.OOO -9,999,999 *^"0,00q,000-S9,999 J 999'\ 



Fig. 1.— Relation o falcohol titration to the bacterial count of milk. Titrations of 116 samples of raw 
and pasteurized market milk with 90 per cent alcohol. 

titration and the bacterial counts. It may be seen that among the 
116 samples plotted there is a wide range in titration of samples 
with low and high bacterial counts. Some samples with a low count 
show a low titration and others a high titration. Among samples 
with a high count some show a low and others a high titration. 



J— 

^ zoo 

X9.0 

r 



k 2.0 



• • • 



£j0GW&7WAf OF 00C73G/&4/L COCW7S. 



Fig. 2.— Relation of alcohol titration to the bacterial count of milk. Titrations of 116 samples of raw 
and pasteurized market milk with 80 per cent alcohol. 

Our results indicate that there is no definite relation between 
alcohol titration and acidity unless the acidity is more than about 
2.20. This is shown in figure 3, where 116 samples are plotted ac- 
cording to their acidity and titration with 90 per cent alcohol, and 
also in figure 4, where the 80 per cent titrations and acidities are 



THE ALCOHOL TEST IN RELATION TO MILK. 



29 



plotted. The plots show that there is a wide range in the alcohol 
titration at all acidities until they reach about 2.20, after which the 
alcohol titration becomes lower in general as the acidities increase. 
This fact holds true for the small number of samples at these high 



b 



Is 



Fig. 3 























































































1 


















































• • 




% 


• 


































♦. 


1 •• 




:• 






• 
































1 




































• 










' 


• 9 


. 












































































































































i 











/.so 



/£0 



/&o 



/SO 



2.20 



2.30 



Z.fO 2.SO 



^.O 2 JO 

^c/o/rr,. 

—Relation of alcohol titration to the acidity of milk. Titrations of 116 samples of raw and pas- 
teurized market milk with 90 per cent alcohol. 



acidities and would probably have been brought out more clearly if 
we had had a larger number of samples with acidity above 2.20. 

If we were dealing with pure cultures of organisms which influence 
the alcohol test the titration with alcohol might be of value in giving 
an idea of the bacterial numbers, as is shown in Table 24, from the 



At 

/3 
































































































































// 
/O 
9 
& 

7 
6 
S 

/ 

O 




































































































9 






































































, 


























" 


























• 










































. 




' 






• 


































" 


































. 














• 










































• 


. 








































































































I 



/.SO 



/.60 



/.70 



/.<SO 



/.90 



2.20 



2.30 



2.40 2SO 



FlQ. 



2.00 2.ZO 
y4C/0/TV. 

4.— Relation of alcohol titration to acidity of milk. Titrations of 116 samples of raw and pasteur- 
ized market milk with SO per cent alcohol. 



results of experiments in which we used pure cultures of lactic-acid 
and rennet-forming bacteria. In milk, however, we have a varied 
bacterial flora to contend with and we can not see from our results 
that the alcohol titration method is of much greater value than the 
simple alcohol test. 



30 BULLETIN 202, U. S. DEPARTMENT OE AGRICULTURE. 

Table 24. — Alcohol titrations of milk inoculated with pure cultures of bacteria. 













Alcohol titra- 












tion. 


Experi- 
ment. 


Culture. 


Age of 

miik 

culture . 


Acid- 
ity. 


Bacteria 
per cubic 
centimeter. 






90 per 
cent to 


80 per 
cent to 












2c.c.of 


2c.c. of 












milk. 


milk. 


1 


Lactic-acid bac- 


Hours. 






c. c. 


c. c. 




teria 





1.95 


64,000 


3.08 


8.88 






2 
3 


1.95 
1.90 


2.86 
3.14 


9! 13 
9.42 


131,000 






4 


1.95 


120, 000 


2.60 


9.52 






5 


1.95 


361,000 


3.05 


9.40 






6 


2.05 


736,000 


3.05 


8.85 






6£ 


2.07 


1,660,000 


1.85 


7.08 




Rennet-forming 














bacteria 



2 


1.95 
1.90 




3.12 
3.03 


9.02 
9.52 


1,600 






3 


1.95 


70,000 


2.50 


9.48 






4 


2.03 


230,000 


2.44 


9.83 






5 


2.04 


2,850,000 


3.20 


9.90 






6 


2.04 


9,300,000 


2.80 


8.12 






6J 


2.07 


11,100,000 


1.87 


6.18 


2 


Lactic-acid bac- 














teria 





2.00 


25,000 
184,000 


3.33 


12.55 






2 


2.00 


3.58 


13.82 






3 


2.08 


475, 000 


3.52 


11.18 






4 


2.04 


1,710,000 


3.73 


9.85 






5 


2.18 


4,900,000 


2.00 


7.32 






6 


2.26 


8,400,000 


2.00 


5.50 






7 


2.33 


22,500,000 


1.06 


1.79 




Rennet-forming 














bacteria 





2.00 


6,100 


3.32 


13.95 






2 


2.00 


51,500 


3.40 


13.36 






3 


2.00 


234,000 


3.98 


10.52 






4 


2.00 


1,325,000 


3.57 


9.35 






5 


2.00 


1,300,000 


1.56 


8.98 






6 


2.04 


13,000,000 


1.25 


2.93 






7 


2.20 


21,800,000 


0.83 


1.21 



THE ALIZAROL TEST. 

When the alcohol has alizarin added to it to act as an indicator 
for the acidity the alcohol test is known as the alizarol test. This 
name was given to the test by Morres (21). The use of alizarin as 
an indicator for the acidity of milk has been known for a long time, 
but Morres (19) was probably the first to combine the alcohol and 
alizarin test. He pointed out that the alcohol test was of more value 
than the litmus test and that the combination with alizarin was 
better than the combination of litmus and alcohol. Morres (20) 
used a 68 per cent alcohol with 1.2 grams of fresh alizarin paste, or 
0.4 gram of dry alizarin to 1,000 c. c. of alcohol. Two cubic centi- 
meters of this alizarin-alcohol solution are mixed with 2 c. c. of milk, 
the same as in the alcohol test. This author found that from the 
coagulation by alcohol and the color of the alizarin it was possible 
to obtain a picture of the condition of the milk. According to 
Morres (20) the alizarol test shows the following conditions: 

1. Lilac-red color. (Milk titrated 7° acid.) 

(a) With no coagulation. The milk should keep sweet more than 6 hours. 

(6) With fine flaky coagulation. The beginning of rennet production is shown 
here. 

(c) With heavy fiocculent coagulation. This indicates advanced rennet forma- 
tion. 



THE ALCOHOL TEST IN RELATION TO MILK. 31 

2. Pale-red color. (Milk titrated 8° acid.) 

(a) With no coagulation or only very fine coagulation. This shows the beginning 

of lactic-acid fermentation. 
(6) With flaky coagulation. Acid and rennet fermentation is indicated. 
(c) With coagulation with very thick flakes. A mixed fermentation is indicated 

with advanced rennet and the beginning of acid fermentation. 

3. Brownish-red color. (Milk titrated 9° acid.) 

(a) With coagulation with fine flakes. Well-advanced pure acid fermentation is 

indicated. 
(6) With coagulation with thick flakes. A mixed fermentation with advanced 

rennet and strong acid fermentation is indicated, 
(c) With coagulation with very thick flakes. A very advanced rennet production 

and little less important acid fermentation is indicated. 

4. Reddish-brown color. (Milk titrates 10° acid.) 

(a) With flaky coagulation. Advanced pure acid fermentation is indicated. 

(b) With thick flaky coagulation. Advanced acid fermentation and the begin- 

ning of rennet production is indicated. 

(c) With very thick flaky coagulation. A proportional mixed fermentation 

which is well advanced is indicated. 

5. Brown color. (Milk titrates 11° acid.) 

(a) With thick flaky coagulation. Pure acid fermentation is indicated . Milk is 
sour; to be detected by smell. 

(6) With very thick flaky coagulation. Some rennet production and well ad- 
vanced acid fermentation is indicated. 

6. Yellowish-brown color. (Milk titrates 12° acid.) 

(a) With very thick flaky coagulation. Acid fermentation is indicated. Milk 
tastes acid. 

7. Brownish-yellow color. (Milk titrates 14° acid.) 

(a) With very thick flaky coagulation. Sour taste is distinctly noticeable. 

8. Yellow color. (Milk titrates 20° acid.) 

(a) With very thick flaky coagulation. Pure acid fermentation is indicated. 
Milk smells and tastes strongly acid and is near the normal coagulation 
point. 

9. Violet color. (Milk titrates 7° acid.) 

No fermentation is indicated, but the milk is abnormal. 

It can not be disputed that a simple test which will picture con- 
ditions in milk, as claimed by Morres, would be of considerable value. 
But will the alizarol test indicate all that Morres claims ? Devarda 
and Weich (6) in 1913, after working with this test, decided that it 
had no value over the alcohol test. In a later paper Devarda (7) 
draws conclusions as follows : 

1. For market control the alcohol test is satisfactory for the determination of the 
quality of milk. 

2. The assertion of Morres that the alizarol test can show a pure rennet and mixed 
fermentation is without scientific or practical significance. 

3. In a pure lactic fermentation the alizarol test stands close to the acidity in its 
color relation, but for the determination of the keeping quality of milk it is of slight 
significance. 

4. The diagnostic value of the alizarol test is limited to an empirical test for milk, 
principally as to its suitability for cheese making which was already employed by 
Eugling in 1882. 

Thoni (30), in a study of the milk supply of Berne, found that 12 
of 85 samples examined were more or less abnormal, according to 



32 



BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 



the alizarol test. Among the other 73 samples of milk, which ac- 
cording to the alizarol test were normal, there were samples which 
had a high bacterial content and which were abnormal according to 
the leucocyte and other tests. From his results Thoni believes that 
the alizarol test is not sufficiently delicate for use in market-milk 
investigations. However, he believes the test is of value as a quick 
means for detecting udder infection in animals. 

It is evident that there is a diversity of opinion as to the value of 
the alizarol test, and our experiments have not been extensive 
enough for us to form a definite opinion in regard to it. 

We have tried the test on a number of samples of milk and have 
not been able to obtain all the color changes which are described by 
Morres. When the acidity was slightly above normal we found a 
change from lilac red to pale red and brownish red. In one sample 
of milk we increased the acidity by the addition of lactic acid and 
obtained the colors named below. 



Amount of N/10 lactic 
added to 50 c. c. of milk. 


Acidity. 


Color of alizarol test. 




1.85 
2.10 
2.42 
2.73 
3.00 
5.15 


Lilac red. 
Pale red. 
Brownish red. 

Do. 

Do. 

Do. 


2 c. c 




6 c. C 


8C. C 


1.5 c. c. normal acid . . 



From our results we believe that alizarin will show slight changes 
in the acidity when the acidity is low, but that the indicator did not 
seem to be very sensitive to high acidities in milk. Morres (22), in a 
paper in 1913, also states that alizarin is of greatest value in indi- 
cating the first changes in acidity and that the color change is so 
gradual at acidities over 16° that the test is of no particular value. 

In regard to the value of the alizarol test we believe that wherever 
the alcohol test can be considered of value, the addition of an indi- 
cator, such as alizarin, may increase the value of the alcohol test by 
possibly giving additional information as to acidity. 

On account of the complexity of the bacterial fermentations in 
market milk we do not believe that the alizarol test gives any very 
valuable information as to the conditions existing in the milk. 

CONCLUSIONS. 

In conclusion, we wish to point out again that the alcohol test 
must be considered from two standpoints: First, in its relation to 
the milk from a single cow or small herd, and, second, in its relation 
to mixed market milk. 

As to the relation of the alcohol test to milk from a single cow, it 
seems evident from the work of other investigators, which is con- 
firmed to some extent by our results, that a positive 68 per cent 



THE ALCOHOL TEST IN EELATION TO MILK. 33 

alcohol test indicates some change in the milk from its normal con- 
dition. In our opinion the value of the alcohol test with milk from 
a single cow or small herd lies in the fact that it would show that the 
milk was abnormal, and in consequence a careful examination should 
be made of the herd. 

When the relation of the alcohol test to mixed market milk is dis- 
cussed, we must consider it on an entirely different basis. In this 
case the test with 68 per cent alcohol may be positive as a result of 
changes produced in milk through bacterial action. The results of 
our work confirm some of the results of other investigators and show 
that the alcohol test may be positive as a result of the growth in milk 
of lactic-acid and rennet-forming bacteria. When the growth of 
these bacteria has reached a point where the acid or rennet is pro- 
duced in sufficient quantities to affect the casein, a coagulation is 
produced when equal volumes of 68 per cent alcohol and milk are 
mixed. Our results, however, do not show that there is any definite 
relation between the alcohol test and the number of bacteria in milk. 
During an examination of 177 samples of raw milk we found that 20 
samples gave a positive test with 68 per cent alcohol. Of these 20 
samples 8, or 42.1 per cent, contained less than 500,000, and 11, or 
57.9 per cent, more than 500,000 bacteria per cubic centimeter. It 
was also found that 39.4 per cent of 142 samples of milk which gave 
no positive alcohol tests contained over 500,000 bacteria per cubic 
centimeter. That there is no definite relation is probably explained 
by the fact that bacteria may increase in large numbers before there 
is much acid or rennet produced. Consequently, if an alcohol test 
were made during that period there would be a high bacterial content 
and yet not enough change produced in the milk by acid or rennet 
to cause a positive test. Besides this point it must be remembered 
that in market milk there is a bacterial flora representing many dif- 
ferent species, many of which may increase without influencing the 
alcohol test. 

As stated before, generally speaking, when the bacterial fermenta- 
tions have advanced to a point where chemical changes are produced, 
the alcohol test will be positive as a result of lactic or rennet fermen- 
tations, or a mixture of both. In such cases the alizarol test may 
be of more value than the plain alcohol test, so far as it may give 
additional information as to the kind of fermentation. From our 
results it seems evident that the acid-and-rennct fermentations may 
be differentiated by means of neutralization of the acidity by sodium 
hydrate. 

The alcohol titration method according to our tests seems to offer 
no particular advantages over the alcohol test. In a study of 116 
samples we were not able to find any definite relation between the 
alcohol titration and the bacterial count. 



34 BULLETIN 202, U. S. DEPARTMENT OF AGRICULTURE. 

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